We will use the power of complete genome sequences and forward genetics to identify novel genes that allow a bacterial pathogen to colonize systemic organs in murine models. Non-typhoidal Salmonella (NTS) are food-borne bacterial pathogens that cause hundreds of millions of cases of diarrheal disease and hundreds of thousands of deaths due to bacteremia annually worldwide. Salmonella enterica serotype Typhimurium is an appropriate model to determine the requirements for systemic infection by NTS as this serotype causes more cases of NTS bacteremia, in children under the age of 5 and in HIV infected individuals, than any other serotype. The availability of complete genome sequence information, the ease of genetic manipulation, and the availability of numerous animal models make serot. Typhimurium an excellent model system. We have generated a collection of 1030 targeted deletion strains in serot. Typhimurium (approximately 25% of the genome), developed a novel oligonucleotide microarray to assay this collection of mutants, and begun forward genetic screening of this mutant collection both in vitro and in vivo in murine models of systemic colonization. We wish to complete the development of these tools and progress to more comprehensive studies of a subset of genes involved in systemic colonization a process that occurs during NTS bacteremia caused by this organism. We will approach this goal by: 1. Completion of our deletion library in all non-essential genes in Salmonella Typhimurium, 2. Screening our complete deletion collection in murine models to identify new genes involved in systemic colonization, 3. Confirming each mutant as defective for systemic colonization, 4. Characterizing mutants that are defective for systemic colonization for their ability to associate with, invade, and replicate in cultured macrophages. PUBLIC HEALTH RELEVANCE: Salmonella is a leading cause of food borne illness, causing ~1.4 million cases of diarrheal disease per year and is the single most common cause of death from food-borne illnesses associated with viruses, parasites or bacteria in the US primarily in immunocompromised persons. In young children and HIV-infected individuals, non-typhoidal Salmonellae (NTS) frequently cause systemic infection that is associated with high mortality. The rise of AIDS in many parts of the world, notably in sub- Saharan Africa, has resulted in a dramatic increase in the frequency of NTS-associated systemic infections. The work described in this project will provide novel targets for vaccines and treatments for acute systemic disease that results from Salmonella infection. This work will have a direct impact on public health.